JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1982, p. 703-7130095-1137/82/040703-11$02.00/0

Vol. 15, No. 4

Atypical Biogroups of Escherichia coli Found in ClinicalSpecimens and Description of Escherichia hermannii sp. nov.

DON J. BRENNER,'* BETTY R. DAVIS,1 ARNOLD G. STEIGERWALT,' CONRADINE F. RIDDLE,'ALMA C. McWHORTER,' STEPHEN D. ALLEN,2 J. J. FARMER III,1 YOSHIHIKO SAITOH,3 AND G.

RICHARD FANNING4

Enteric Section, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303331;Department of Clinical Pathology, Indiana University Medical College, Indianapolis, Indiana 462022; ClinicalMicrobiology Branch, Central Clinical Laboratory, Tohoku University School of Medicine, Sendai, Japan3;

and Division ofBiochemistry, Walter Reed Army Institute of Research, Washington, DC 200124

Received 16 November 1981/Accepted 16 December 1981

DNA relatedness was used to define the biochemical boundaries of Escherichiacoli. A large number of biochemically atypical strains were shown to belong tobiogroups of E. coli. These included strains negative in reactions for indole, allthree decarboxylases, D-mannitol, lactose, or methyl red and strains positive inreactions for H2S, urea, citrate, KCN, adonitol, myo-inositol, or phenylalaninedeaminase. Frequency and source data are presented for these atypical E. colibiogroups. One group of KCN-positive, cellobiose-positive, yellow-pigmentedstrains was 84 to 91% interrelated but only 35 to 45% related to E. coli. The name

Escherichia hermannii sp. nov. is proposed for this group of organisms that was

formerly called Enteric Group 11 by the Enteric Section, Centers for DiseaseControl, Atlanta, Ga. Twenty-nine strains of E. hermannii have been isolated inthe United States from a variety of clinical sources, principally wounds, sputum,and stools. Three additional strains were isolated from food. E. hermannii strainsare gram-negative, oxidase-negative, fermentative, motile rods. In addition toyellow pigment and positive KCN and cellobiose tests, the biochemical reactionscharacteristic of 32 strains of E. hermannii were as follows: gas from D-glucose,acid from D-glucose, maltose, D-xylose, L-arabinose, L-rhamnose, and D-manni-tol; no acid from adonitol or inositol; variable acid production from lactose andsucrose; positive tests for indole, methyl red, and mucate; negative tests forVoges-Proskauer, Simmons citrate, H2S, urea, phenylalanine deaminase, andgelatin hydrolysis; negative or delayed test for L-lysine decarboxylase andnegative test for L-arginine dihydrolase; and positive test for ornithine decarbox-ylase. E. hermannii strains were resistant to penicillin, ampicillin, and carbenicil-lin and sensitive to other commonly used antibiotics. Wounds account for almost50% of human isolates of E. hermannii, followed by sputum or lung isolates (ca.25%) and stool isolates (20%).

Escherichia coli was among the first species tobe defined and separated from other species onthe basis of DNA relatedness (2, 3, 5, 6). Thestrains were studied by intraspecies DNA relat-edness, genome size, and guanine-plus-cytosine(G+C) content. They were chosen on the basesof geographic origin, source (animal, human,environment), site of isolation (intestinal, ex-traintestinal), pathogenicity, and serology. Withone exception, the Alkalescens-Dispar biogroup(anaerogenic, lactose-negative, nonmotilestrains), biochemical profiles, and specific atypi-cal reactions were not considered in choosingthe strains used in these studies.During the past decade several E. coli-like

biogroups have been identified. Some, such asH2S-positive (10, 14, 15), urea-positive (16, 18,

21, 22), and citrate-positive (19, 23) biogroups,are assumed to be E. coli. Other less familiarbiogroups include strains positive in reactionsfor KCN, adonitol, inositol, or phenylalaninedeaminase and strains negative in reactions formethyl red or mannitol. With the exceptions ofH2S-positive (10) and urea-positive (16, 21, 22)biogroups, little is known about the source,relative frequency, and potential clinical signifi-cance of these E. coli-like biogroups.Using DNA hybridization, we showed that

many of the atypical strains are E. coli. Bio-chemistry, frequency, source, and, where avail-able, clinical diagnosis are presented for each ofthese biogroups. A group of yellow-pigmented,KCN-positive, cellobiose-positive strains, for-merly called Enteric Group 11 by the Enteric

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TABLE 1. DNA relatedness of E. coli K-12 to various E. coli-like biogroupsSource of Atypical biochemical RBR, RBR,

unlabeled DNA reaction(s)' 600C (%)b D (%c" 750C (%)h

K-121087-751100-751552-74A183a1223-74E103824-74931-752916-731644-754909-73561-75354-742030-751902-752140-74573-751665-751582-75558-75571-752431-741664-754621-74478-753757-731354-74799-751470-751471-752154-752465-735106-72426-733216-74134-75308-75757-752399-742397-74589-751943-742302-743292-74907-754367-731892-74483-74A433a526-751555-752128-7946-802103-79689-753270-742132-733949-73F1119-41E77aU7-41

Ado'Ado'Cit+Cit+Cit+Cit+Cit+, H2S+Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-Dec-H2S+H2S+H2S+H2S+H2S+H,S+, Ind-H2S+, Lac-Lac-Ino+Ino+Ino+, H2S+Ino+, Ind-Man-Man-Man-Man-Man-Man-, H2S+Mr-, Man-Mr-, Man-Mr-, Man-PPA+PPA+PPA +

1001001001001009795100100100100100100100100100100100100100100999999989898979797979696969595938910010010010010010096929390941009493

991009691100100100

0.00.00.00.0

1001001001001009195100100100100100100999999979695959310010098104949296949493989796908991891001001009998100958993941001008985969384100100979110010089

0.5

0.5

0.0

1.01.50.5

1.00.0

0.0

0.00.0

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DESCRIPTION OF E. HERMANNII SP. NOV.

TABLE 1-ContinuedSource of Atypical biochemical RBR, RBRunlabeled DNA reaction(s)a 6OoC (%)b D 75%)'RbR

(E. coli K-12) 75°C (%)b

5079-71 Ure+ 100 1005330-56 Ure+ 100 1004574-72 Ure+ 100 0.5 100708-74 Ure+ 100 975143-71 Ure+ 100 972110-75 Ure+ 99 99759-70 Ure+ 95 965539-71 Ure+ 78 7.0 60U14-41 KCN+ 100 0.0 100F1961 KCN+ 100 100899-73 KCN+, Cel+, YP+ 46 14.5 131933-74 KCN+, Cel+, YP+ 45 12.0 17980-72 KCN+, Cel+, YP+ 44 14.5 152472-72 KCN+, Cel+, YP+ 40 14.5 143300-74 KCN+, Cel+, YP+ 40 13.0 12a Ado, adonitol; Cit, Simmons citrate; Dec, arginine dihydrolase, lysine decarboxylase, and ornithine

decarboxylase; Ind, indole; Lac, lactose; Ino, inositol; Man, mannitol; MR, methyl red; PPA, phenylalaninedeaminase; Ure, urease; Cel, cellobiose; YP, yellow pigment; +, positive reaction in 1 to 7 days; -, negativereaction.

b RBR, Relative binding ratio = percent (heterologous DNA bound to hydroxyapatite/homologous DNAbound to hydroxyapatite) x100.

c D, Percent divergence. D was calculated on the assumption that a 1°C decrease in the thermal stability of aheterologous DNA duplex compared with that of the homologous DNA duplex is caused by each 1% of unpairedbases within the duplex. D values are given to the nearest 0.5%.

Section, Centers for Disease Control (CDC)(unpublished data), was shown to represent anew species in the genus Escherichia. The nameEscherichia hermannii sp. nov. is proposed forthis organism.

MATERIALS AND METHODSBacterial strains. The 76 strains of E. coli and 8

strains of E. hermannii used in DNA relatednessstudies are listed in Tables 1 and 2. Biochemical andsource data were obtained from a much larger numberof strains that are not listed individually. Data onorigin, source, location, clinical diagnosis, associatedillness, and age were taken directly from a computerrecord based on the original information furnishedwith the culture. These data were not further verified.All E. coli and E. hermannii strains were sent to CDCfrom state and federal laboratories between 1971 and1980.Nomenclature. With the exception of "Citrobacter

amalonaticus" (Levinea amalonatica), all names usedhave standing in nomenclature (20). The classificationused in the eighth edition of Bergey's Manual ofDeterminative Bacteriology was used (19), with thefollowing exceptions: Escherichia blattae, Entero-bacter sakazakii, Enterobacter gergoviae, Klebsiellaoxytoca, and Yersinia ruckeri, which were not includ-ed in the eighth edition. Some, but not all, of thestrains that we called Enterobacter agglomerans areundoubtedly synonymous to Erwinia stewartii, Er-winia herbicola, and Erwinia uredovora. Since wecould not identify the synonymous strains, and sincethe thrust of this paper was clinical and these strains

were used as controls, there were all called Entero-bacter agglomerans. The names Citrobacter diversus,Citrobacter amalonaticus, Morganella morganii, Pro-videncia rettgeri, Providencia alcalifaciens, and Pro-videncia stuartii were used rather than Citrobacterintermedius biotype b, Citrobacter intermedius bio-type a, Proteus morganii, Proteus rettgeri, Proteusinconstans subgroup A, and Proteus inconstans sub-group B, respectively (the latter names appear in theeighth edition of Bergey's Manual of DeterminativeBacteriology [9]).

Media, biochemical tests, and antimicrobial agentsusceptibility tests. Strains used in DNA relatednessstudies were maintained on blood agar base or meatextract agar. Unlabeled DNA was prepared from cellsgrown to stationary phase in 1.5 liters of brain heartinfusion broth on a dry-air rotary shaker at 37°C. Themedium we used for labeling cells with 32PO4 and themedia and reaction conditions for biochemical testshave previously been described (4, 11, 13). Antibio-grams were done on Mueller-Hinton agar by the diskmethod of Bauer et al. (1), as previously described(12).G+C content. The G+C content of DNA was deter-

mined optically by thermal denaturation (17).DNA preparation and DNA reassociation. The meth-

ods used for DNA preparation and reassociation havebeen previously described (7). All DNA reassociationreactions were done at least twice. Binding of homolo-gous DNA to hydroxyapatite was between 60 and 75%before normalization. Control reactions containingonly labeled DNA showed less than 5% binding tohydroxyapatite. The control values were subtractedfrom heterologous reaction values before normaliza-tion.

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706 BRENNER ET AL.

TABLE 2. Intra- and interspecies DNA relatedness of E. hermannii

Source of unlabeled DNA RBR, 600C D (%)b RBR, 750C

Escherichia hermanii 980-72c 100 0.0 100E. hermannii 1933-74 91 1.0 92E. hermannii 899-73 91 1.0 90E. hermannii 2868-70 88 0.5 90E. hermannii 2472-72 88 0.5 88E. hermannii 3300-74 87 1.0 89E. hermannii 4045-67 86 1.0 88E. hermannii 4408-70 84 0.5 87Escherichia coli 614-41 (KCN+)d 42 16.0E. coli 1003-68 (KCN+) 38E. coli 708-74 (Ure+) 40 15.5 12E. coli 5143-71 (Ure+) 38E. coli 571-75 (Dec-) 37 7E. coli K-12 31 13.5Escherichia blattae 9005-74 32Salmonella typhimurium LT2 39Citrobacterfreundii 460-61 40Citrobacter diversus 1381-70 41 13.5 9Citrobacter amalonaticus 25406 41 13.5 8Klebsiella pneumoniae 2 41Enterobacter aerogenes 1627-66 35Enterobacter cloacae 1347-71 46Enterobacter sakazakii 4562-70 44Enterobacter agglomerans 6003-71 50 14E. agglomerans 4388-71 48 16.0 13E. agglomerans 5378-71 48E. agglomerans 219-71 42E. agglomerans 1600-71 42E. agglomerans 1429-71 41E. agglomerans 2780-70 37E. agglomerans 5422-69 36E. agglomerans 3123-70 35E. agglomerans 1741-71 33E. agglomerans 6070-69 33E. agglomerans 23372 30E. agglomerans 3482-71 29 16.0Hafnia alvei 5632 23 17.0H. alvei 4510 20 17.0Serratia marcescens 868-57 21Erwinia amylovora EA 178 26Erwinia salicis ES 102 26Erwinia quercina EQ 102 24Erwinia chrysanthemi SR-32 24Erwinia cypripedii EC 155 23Erwinia tracheiphila 27004 22Erwinia carotovora 495 22Yersinia enterocolitica 497-70 23Yersinia ruckeri 23Morganella morganii 25830 25Providencia alcalifaciens 3370-67 13Providencia rettgeri 1163 12Providencia stuartii 3233-68 9Proteus mirabilis PM-1 7Proteus vulgaris PR-1 7

a See footnote b, Table 1.b See footnote c, Table 1.c Strain 980-72 was labeled.d See footnote a, Table 1, for reaction abbreviations.

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DESCRIPTION OF E. HERMANNII SP. NOV.

RESULTSDNA relatedness. DNA from E. coli K-12,

used in previous DNA relatedness studies (2, 3,5, 6), was labeled with 32P04 and reacted withunlabeled DNAs from strains representing eachof the E. coli-like biogroups of interest. All butone of the biogroups tested were 90 to 100%related to E. coli (Table 1) and are thereforeconsidered as biochemically unusual strains ofE. coli (biogroups) rather than separate species.The exceptions were five strains that were posi-tive in reactions for KCN and cellobiose andproduced yellow pigment. Strains that were onlyKCN positive (Table 1) or that only producedyellow pigment (data not shown) were E. coli.The KCN-positive, cellobiose-positive, yellow-pigmented strains were 40 to 45% related to E.coli K-12 in 60°C reactions, and the relatedsequences contained some 14% divergence (un-paired bases). In 75°C reactions relatedness toE. coli fell to about 15%. In contrast, with onlyone E. coli strain (5539-71) did related sequencescontain more than 1.5% unpaired bases or didrelatedness with E. coli K-12 in 75°C reactionsfall below 80% (Table 1).DNA from KCN-positive, cellobiose-positive,

yellow-pigmented strain 980-72 (subsequentlydesignated as the type strain) was labeled with32P04 and tested for relatedness to similarstrains, to various E. coli biogroups, and torepresentative species of Enterobacteriaceae(Table 2). Seven other KCN-positive, cellobi-ose-positive, yellow-pigmented strains were

85% or more related to strain 980-72 in 60°Creactions. The related sequences contained1.5% or less divergence, and relatedness re-

mained above 85% in reactions done at 75°C. InDNA reactions with other Enterobacteriaceae,strain 980-72 was 30 to 42% related to E. coli andE. blattae. A similar level of relatedness (35 to46%) was demonstrated to species of Citro-bacter, Salmonella, Klebsiella, and Entero-bacter species other than E. agglomerans. Re-latedness to strains representing the variousDNA relatedness groups in the Enterobacteragglomerans complex ranged from 29 to 50%.Divergence between strain 980-72 and all ofthese genera was 13 to 16%, and relatedness fellto below 15% in 75°C reactions. Strain 980-72was 20 to 25% related to Serratia, Hafnia,Erwinia, Yersinia, and Morganella species andabout 10% related to species of Proteus andProvidencia. The new species E. hermannii was

defined on the basis of these data.G+C content. The G+C contents of DNAs

from eight strains of E. hermannii were deter-mined at least twice spectrophotometrically, bythermal denaturation. The ratios obtained were

between 53 and 58 mol% G+C, well within therange for Enterobacteriaceae.

Biochemical reactions and description of E.hermannii. Biochemical test reactions for 32 E.hermannii strains and for the type strain areshown in Table 3. E. hermannii is a gram-negative, oxidase-negative, catalase-positive,nonsporeforming rod, motile with lophotrichous(two or more flagella at one or both poles) ordegenerately peritrichous (flagella distributedunevenly over much of the cell periphery) flagel-la (Fig. 1), that reduces nitrate to nitrite, fer-ments D-glucose and other carbohydrates withthe production of acid and gas, has 53 to 58mol% (thermal denaturation) G+C in its DNA,and is isolated from food and a variety of humanclinical specimens. It is positive in reactions formaltose, D-xylose, L-arabinose, L-rhamnose, D-mannitol, mucate, indole, methyl red, and orni-thine decarboxylase, negative in reactions foradonitol, myo-inositol, Voges-Proskauer, Sim-mons citrate, H2S, urea, phenylalanine deami-nase, arginine dihydrolase, and gelatin, negativeor delayed for lysine decarboxylase, and vari-able in reactions for lactose and sucrose. Afurther description of E. hermannii is found inthe tables and figure, as well as below. The typestrain (holotype) ATCC 33650 (CDC 980-72) wasisolated from an infected toe of a 17-year-oldfemale in Louisiana. We propose Escherichiahermannii as a new species. It is treated as amodem Latin noun hermannii (her * man' ni * ipronounced her * mahn' nee * eye), of Hermann;in honor of George J. Hermann, former chief ofthe Enteric Section at the CDC, for his manycontributions to enteric bacteriology and forLloyd G. Herman, formerly of the Environmen-tal Services Branch, National Institutes ofHealth, Bethesda, Md., for his contributions tothe study of yellow-pigmented bacteria.

Differentiation of E. hermannii from other En-terobacteriaceae. Table 4 shows tests that areuseful in identifying E. hermannii. Yellow pig-ment alone separates E. hermannii from allEnterobacteriaceae except for certain species ofEnterobacter and an occasional E. coli strain.Positive KCN, cellobiose, and motility testsseparate E. hermannii from shigellae. Positivemotility and ornithine decarboxylase and nega-tive adonitol and D-sorbitol tests differentiate E.hermannii from klebsiellae. It is distinguishedfrom Enterobacter species on the basis of itspositive indole and negative Voges-Proskauerreactions. Other tests helpful in separating E.hermannii from individual Enterobacter speciesare methyl red, Simmons citrate, decarboxyl-ases, and malonate. C. diversus is KCN negativeand positive in tests for adonitol, malonate,urease, D-sorbitol, and Simmons citrate, all ofwhich distinguish it from E. hermannii; a nega-tive indole test and positive Simmons citrate andD-sorbitol tests differentiate C. freundii from E.

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708 BRENNER ET AL.

TABLE 3. Biochemical reactions of E. hermannii and of atypical E. coli biogroups"E. hermannii % Positive in given E. coli biogroup'

TeStb 32 Type 1otTIstrains| strain KCN+ Cel+ MR- Cit+ Ure+ Dec- MoGa(%poii (ATCC (39) (68) (42) (52) (48) (209) (380)

positive) 33650)___ __ ____1 1TIndole 100 + 77 72 81 92 98 72 (1) 76Methyl red 100 + 95 (5) 100 0 100 100 92 (2) 92 (2)Voges-Proskauer 0 - 0 0 0 0 0 0 0Citrate, Simmons 0 (19) - 0 (8) 0 (2) 0 10 (90) 0 1 (1) 0 (2)H2S on TSI 0 - 3 11 0 0 0 1 1Urease, Christensen 0 - 0 3 0 0 94 (6) 1 0Phenylalanine deaminase 0 - 3 0 0 0 2 1 2Lysine decarboxylase 6 (34) (+) 67 (8) 84 (2) 59 89 (2) 88 0 39 (6)Arginine dihydrolase 0 _ 5 (29) 13 (22) 10 (2) 11 (25) 17 (52) 0 4 (20)Ornithine decarboxylase 100 + 32 (18) 64 (5) 29 (2) 42 (9) 67 (8) 0 24 (9)Motility 100 + 42 66 24 65 65 (1) 18 (1) 0Gelatin liquefaction (22°C) 0 - 0 0 0 0 0 0 0KCN, growth in 94 (6) + 49 (51) 0 0 7 0 0 0 (2)Malonate utilization 3 (13) - 0 (5) 0 (2) 0 0 0 0 0D-Glucose, acid 100 + 97 (3) 100 95 (5) 100 100 100 100D-Glucose, gas 97 + 74 94 36 (10) 73 94 40 0Lactose 44 (16) - 79 (8) 73 (15) 68 (7) 80 (9) 71 (4) 46 (12) 37 (13)Sucrose 44 - 32 29 (6) 32 (7) 25 (3) 35 13 (5) 16 (4)D-Mannitol 100 + 95 (3) 100 69 (12) 94 92 93 (1) 91 (2)Dulcitol 19 (75) (+) 50 (8) 59 (6) 7 (10) 72 (4) 63 (10) 30 (11) 35 (11)Salicin 41 (41) _ 24 (11) 29 (34) 5 (10) 36 (10) 26 (15) 22 (11) 15 (16)Adonitol 0 - 23 2 2 0 2 7 (1) 5myo-Inositol 0 _ 3 0 0 0 2 1 0D-Sorbitol 0 - 95 (5) 89 (8) 42 (29) 90 (5) 81 (6) 77 (13) 74 (14)L-Arabinose 100 + 97 (3) 96 (4) 85 (12) 97 (3) 100 87 (6) 89 (6)Raffinose 41 _ 29 (8) 44 20 (10) 31 38 (4) 15 (2) 15 (3)L-Rhamnose 97 (3) + 68 (8) 84 (2) 85 (5) 66 (10) 81 (2) 65 (12) 68 (17)Maltose 100 + 97 (3) 97 (3) 76 (14) 100 100 82 (9) 80 (12)D-Xylose 100 + 92 (3) 94 83 (15) 100 92 79 (4) 74 (7)Trehalose 100 + 100 96 62 (38) 93 98 89 (7) 91 (7)Cellobiose 97 (3) + 0 46(54) 0 0 2 (2) 2 (1) 1a-Methyl-D-glucoside 0 _ 0 0 0 0 0 0 0Esculin hydrolysis 41 (41) (+) 26 (5) 30 (30) 0 (8) 25 (7) 25 (13) 12 (10) 5 (12)Melibiose 0 - 85 83 25 (32) 79 (11) 68 (5) 43 (7) 40 (11)D-Arabitol 8 - 30 0 (4) 4 0 5 12 (1) 6Mucate 97 (3) + 79 (5) 83 39 (7) 74 (3) 71 34 (4) 29 (8)Lipase, corn oil 0 - 0 0 0 0 2 0 0DNase (25°C) 0 _ 0 0 0 0 0 0 0NO3+ -* N02 100 + 97 100 93 100 100 99 98Oxidase, Kovacs 0 - 0 0 0 0 0 0 0ONPG (,-galactosidase) 100 + 82 (11) 83 (4) 68 (11) 80 73 (18) 48 (20) 46 (20)Yellow pigment 100 + 0 4 0 0 0 0 0D-Mannose 100 + 100 100 79 (22) 91 (5) 100 95 (5) 97 (3)Erythritol 0 _ 0 0 0 (3) 0 0 1 0Glycerol 3 (62) - 87 (11) 64 (24) 20 (43) 76 (17) 60 (27) 69 (17) 63 (24)Jordan tartrate 34 - 87 (3) 94 71 (7) 86 98 86 (3) 83 (4)Acetate 78 (13) + 62 79 (4) 22 (7) 90 81 (8) 39 (9) 37 (5)Citrate, Christensen 61 (16) (+) 16 (34) 25 (30) 0 (29) 41 (41) 26 (36) 14 (17) 18 (25)H2Sinpeptone iron agar 0 4 10 0 0 0 0 1

a The values given are for 48-h incubation (except for oxidase) at 36 11C unless otherwise indicated. Thevalues in parentheses are delayed reactions that became positive between 3 and 7 days. +, Positive reactionwithin 48 h; (+), positive reaction in 3 to 7 days; -, negative reaction after 7 days.

b TSI, Triple sugar iron; ONPG, o-nitrophenyl-,-D-galactopyranoside; PIA, .

'Values in parentheses for E. coli biogroups are the numbers of strains tested. Cel, Cellobiose; MR, methylred; Cit, Simmons citrate; Dec, lysine decarboxylase, arginine dihydrolase, and ornithine decarboxylase; Mot,motility; Lac, lactose; Man, D-mannitol; Ado, adonitol; Ino, myo-inositol; Mal, maltQse; Xyl, xylose; Ara, L-arabinose; Tre, trehalose; Nit, nitrate reduction; Phe, phenylalanine; +, positive; -, negative.

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VOL.15,1982~~DESCRIPTIONOF E. HERMANNII SP. NOV. 70

Lac-(414(

749701 (1)6 (1)31

52 (4)7 (26)

45 (4)27 (1)01(10 (1)

10053013 (4)90 (1)44 (9)27 (14)4

80 (9)94 (2)10 (3)70 (14)91 (5)77 (3)94 (4)2 (1)0

20 (13)34 (12)4

47 (5)00

99014 (29)0

100070 (20)90 (1)55 (7)21 (28)7

Man-(80)

819001 (3)

333 (10)9 (51)

62 (3)22 (1)00 (1)0 (1)

96 (4)58 (5)39 (13)130

15 (7)16 (12)07

56 (10)9716 (1)69 (15)91 (5)73 (7)79 (13)0013 (10)42 (10)

46 (6)00

1000

55 (17)0

82 (16)0

53 (26)8944 (15)24 (39)2

Ado-(178)

8899 (1)00

81 (2)6 (212)

29 (13)59 (1)02 (6)0

1008676 (8)15 (1)99 (1)4 (1)

61(14)97 (3)0

90 (6)97 (2)18(3)81(7)91(7)94 (1)95 (5)

044 (19)76 (9)94 (2)72(2)00

98(1)0

76 (9)0

1000

79 (15)90 (2)55 (3)10 (2)6)7

TABLE 3-Conhitiuled% Positive in given E. co/i biogroup'

Ino(19)

8410000

29S0

5811(16)58 (5)5300 (5)0

10090 (5)68 (16)427447 (23)42 (11)0

95 (5)798842 (5)83 (6)95 (5)60 (20)9300

38 (13)89116700

1000

1000

100081 (3)888219 (19)38

Mal-(47)

558900

00

434 (17)

32 (6)13(2)000

1004351 (17)3083 (1)28 (17)9 (19)40

60 (15)73 (16)19 (4)67 (20)0

58 (16)74 (16)000 (11)

35 (27)0

39 (4)00

1000

73 (12)0

1000

38 (29)71 (7)19 (4)12 (19)3

(130))

6697 (2)002 (1)30

39 (2)5 (20)

49 (6)

00 (2)0

10049 (2)35 (11)2.2 (5)87 (2)28 (2)12 (13)3

710)90 (6)23 (5)62 (10)83 (8)0

93 (4)0 (2)03 (15)

37 (9)3

40 (5)00

990

46 (18)0

1000

56 (30)82) (2)31 (11)19 (23)5

Ara(29)

6693 (4)00000

243 (17)

28 (7)14000

10031 (4)48 (3)24 (14)9028 (14)14 (21)37

83 (7)

36 (7)64 (11)56 (26)70 (7)76 (16)000 (8)

62 (8)8

24 (4)00

1000

540

1000

46 (27)791520 (8)4

Tre-(21)

67

00 (10)

14

48 (5)0 (32)

37380

0

1005758 11)30703715(10)05

6379 (ii)21(5S)68 (5)74 (5)790100

1650 (20)0 (10)

3700

1000

78 (22)0

60 (20)S

32 (21)68 (5)44 (11)35 (6)15

Nit(9)

6767 (11)

0000

67 (22)00 (11)

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hermannii; and C. amalonaticus is separablefrom E. hermannii on the basis of its positivetests for Simmons citrate, urease, arginine dihy-drolase, and sorbitol. Positive KCN and cellobi-ose tests and a negative D-sorbitol test separateE. her-mannii from E. coli.

Antimicrobial susceptibility. By disk diffusionall but one E. her,inannii strain (97%) wereresistant (including zone sizes intermediate be-

tween sensitive and resistant) to penicillin, am-picillin, and carbenicillin and sensitive to othercommonly used antibiotics (Table 5). Excep-tions were strain 472-81, which was sensitive tothese r3-lactam antibiotics, and strain 470-81,which was also resistant to streptomycin andtetracycline.

Origin, source, and clinical information. The32 E. hermannii strains were isolated in 12

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710 BRENNER ET AL.

FIG. 1. E. hermannii cells showing flagella x 1.000.

states, most of which were located on the WestCoast or East Coast of the United States. Whensex was given, 11 of 15 strains were isolatedfrom men. One isolate was from a patient of lessthan 5 years of age, two were from adultsbetween 20 and 49 years of age, and one wasfrom an adult of more than 50 years of age. Noother age data were given. Twenty-five of 28 E.hermannii strains for which sources were knownwere isolated from humans. Twelve of thesewere isolated from wounds; six, from sputum orlung; five, from stools; and one each, from bloodand spinal fluid. There were no isolates from theurinary tract or from the female reproductivetract.

Atypical E. coli biogroups. Biochemical reac-tions for a number of atypical E. coli biogroupsare shown in Table 3. Some of these biogroups(lactose negative; nonmotile, anaerogenic; de-carboxylase negative) are already recognizedand have been identified by many clinical labo-ratories. Others (phenylalanine deaminase, ci-trate, KCN, mannitol, adonitol, inositol, or ure-ase positive) are not well known and presentmost laboratories with problems in identifica-tion. Each of these biogroups can be furthersubdivided on the basis of carbohydrate fermen-tation patterns and decarboxylase reactions.Furthermore, these biogroups are not mutuallyexclusive, so combinations of atypical reactionsare seen. Most biogroups contain some strainsnegative in reactions for indole, methyl red,arabinose, maltose, xylose, or trehalose andsome strains positive for citrate, H2S, urease,phenylalanine deaminase, KCN, adonitol, orinositol. Those shown in Table 3 are used toexemplify, rather than comprehensively de-scribe, biochemically atypical E. c oli biogroups.The atypical E. coli biogroups in our collec-

tion were isolated from every state in the UnitedStates, Guam, and Puerto Rico, as well as from20 foreign countries. Stool and urinary tractwere by far the most frequent sites of isolationfor the atypical E. c/oli strains (47 and 39%,respectively). Seven percent of total isolateswere from blood, 3% were from wounds, 2%were from sputum and lung, and lower percent-ages were from vagina and cervix and fromspinal fluid. The most notable exception to theoverall source trend was in the methyl red-

negative biogroup, in which 30 isolates werefrom the urinary tract, 3 were from blood, andonly 1 was from a stool specimen. In contrast toE. hermannii, the E. coli biogroups were isolat-ed more frequently from women (61% of 921strains). Many of the biogroups are isolatedmost frequently from people less than 5 years ofage or above the age of 50. Few isolates wereobtained from people between the ages of 6 and19 years.

DISCUSSIONThe family Enterobacteriaceae contains spe-

cies such as Edw-ardsiella tacda and Yer.sinziapseiudotiubercullosis that are very similar bio-chemically and others, including Yersinia enter-ocolitica and E. coli, that are biochemicallyheterogeneous. The original object of this studywas to define the biochemical limits of E. coli.This was done by choosing a number of bio-chemically atypical, E. coli-like strains and de-termining their DNA relatedness to typical E.coli. E. hermannii was discovered as a groupthat showed 40 to 46% relatedness to E. coli K-12. The type strain of E. hermnannii was mostclosely related (35 to 50%) to species of Entiero-bacter, Klebsiella, Citrobacter, Sallmonella, andEscherichia. These are the "core" genera of thefamily Enterobacteriaceae, having 40 to 50%intergeneric DNA relatedness. Relatedness oflabeled E. h/ermnannii 980-72 to E. coli K-12 was31%, significantly lower than the 44% related-ness obtained in the reciprocal reaction (labeledE. coli K-12 DNA reacted with unlabeled E.hermannii 980-72 DNA). This was probably dueto the relatively small genome size of E. coli K-12 compared with other E. coli strains (3, 5).This assumption is supported by the 37 to 42%relatedness of E. her,mannii 980-72 to other E.coli strains (Table 2).

It was previously argued that overall DNArelatedness was the single most important crite-rion for determining and defining taxa at thespecies level (7, 8). DNA relatedness and pheno-typic data leave no doubt that E. hermanniii is anew species. It was previously argued (7, 8) thatphenotypic characteristics should take prece-dence over DNA relatedness in assigning aspecies to a given genus. On the sole basis ofDNA relatedness, E. hermannii could have beenassigned to Enterobacter, or almost as easily toCitrobacter, Klebsiella, Escherichia, or evenSalmonella. E. hermannii is biochemically mostsimilar to C. (Levinea) amalonaticus and E. coli(Table 4). When phenotypic characteristics areweighed, the choices are Escherichia or Citr-o-bacter or creation of a new genus intermediatebetween these two genera.

E. h/ermannii is KCN positive and cellobiosepositive and produces a yellow pigment. These

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TABLE 5. Antimicrobial susceptibility pattern of 32E. hermannii strains'

Zone diam (mm) No. ofAntimicrobial agent

Range Mean sensitive strains

Colistin 10-16 14 31Nalidixic acid 22-26 24 32Sulfadiazine 17-28 22 32Gentamicin 23-26 24 32Streptomycin 9-20 18 31Kanamycin 14-26 23 31Tetracycline 6-23 21 31Chloramphenicol 16-29 20 30Penicillin 6-14 7 1Ampicillin 6-24 12 1Carbenicillin 6-29 12 1Cephalothin 22-27 25 32

a A zone size intermediate between that accepted assensitive and that accepted as resistant was shown bysingle strains to colistin, kanamycin, and penicillin andby two strains to chloramphenicol.

reactions distinguish it from E. coli. Other E.hermannii reactions that differ from E. coli are19% delayed positive Simmons citrate, 6% posi-tive (34% delayed positive) lysine decarboxyl-ase, and negative D-sorbitol. Yellow pigmentand negative reactions for D-sorbitol, argininedihydrolase, urease, and Simmons citrate (19%delayed positive) separate E. hermannii from C.amalonaticlis and from other Citrobacter spe-cies. Other reactions of value in distinguishingE. hermannii from C. amcalonaticuis are lysinedecarboxylase (40% positive or delayed positivein E. hermannii and no known positive strains inany Citrobacter species) and raffinose (41% pos-itive in E. hermannii, negative in C. amalonati-cus).

It is difficult to think of citrate- and urease-negative, arginine-negative, yellow-pigmentedstrains that are frequently lysine positive asmembers of Citrobacter. Certain E. coli strainsare known to be KCN positive, cellobiose posi-tive, or D-sorbitol negative (7%) or to produce ayellow pigment. The new organism was there-fore assigned to the genus Escherichia. Otherspecies including indole-positive biogroups ofthe Enterobacter agglomerans complex, Entero-bacter sakazakii and Escherichia adecarboxy-lata (not included in this study), are phenotypi-cally similar to E. hermannii. Further study ofthese organisms may result in the proposal of anew genus.The E. hermannii strains we studied were

most often isolated from wounds and frequentlyfrom sputa and stools. These sources of isolationdiffer from those of E. coli, which is mostfrequently isolated from stools, the urinarytract, and, less frequently, blood. Assuming thatthe CDC sample represents the frequency with

which E. hermannii is isolated in the UnitedStates, we believe it is a rare species which maycause human disease. A retrospective study ofE. hermannii as well as prospective studies ofnew isolates will be necessary to determine itstrue incidence and clinical significance.

It was conclusively shown that all other atypi-cal, E. coli-like strains studied were biogroups ofE. coli. It is possible that the atypical positivereactions (KCN, cellobiose, citrate, urease,adonitol, inositol, phenylalanine deaminase) aremediated by plasmids. This was previouslyproven to be the case for H2S-positive (14, 15),urease-positive (21), and citrate-positive (19)strains. Neither the presence of plasmid-mediat-ed genes nor the absence of reactions normallyassociated with E. coli should prevent the identi-fication of most E. coli strains. Atypical strainsare identifiable on the basis of their overallbiochemical profile. Certainly no laboratoryshould hesitate to report as E. coli otherwisetypical strains that are negative in reactions forindole, lactose, gas from glucose, arabinose,trehalose, xylose, maltose, nitrate reduction,mannitol, motility, or even methyl red or other-wise typical strains positive in reactions for H,S,KCN, urease, citrate, adonitol, inositol, or cel-lobiose. Admittedly, strains with more than oneatypical reaction pose a problem for identifica-tion. It is advisable to have strains with multipleaberrant reactions confirmed by a referencelaboratory.

Despite having more than 2,000 atypical E.coli strains in our collection, little is reallyknown about their epidemiology. Source infor-mation indicates that 85% of most atypical bio-groups are isolated from stools or the urinarytract, with most of the remaining isolates fromblood and wounds. One notable exception ap-pears to be the methyl red-negative strains iso-lated almost exclusively from the urinary tract.

LITERATURE CITED

1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M.Turck. 1966. Antibiotic susceptibility testing by a stan-dardized single disk method. Am. J. Clin. Pathol. 45:493-496.

2. Brenner, D. J. 1970. Deoxyribonucleic acid divergence inEnterobacteriaceae. Dev. Ind. Microbiol. 11:139-153.

3. Brenner, D. J., and S. Falkow. 1971. Molecular relation-ships among members of the Enterobacteriaceae. Adv.Genet. 16:81-118.

4. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. V.Citarella, and S. Falkow. 1969. Polynucleotide sequencerelationships among members of Enterobacteriaceae. J.Bacteriol. 98:637-650.

5. Brenner, D. J., G. R. Fanning, F. J. Skerman, and S.Falkow. 1972. Polynucleotide sequence divergence amongstrains of Escherichia (oli and closely related organisms.J. Bacteriol. 109:953-965.

6. Brenner, D. J., G. R. Fanning, A. G. Steigerwalt, I. 0rs-kov, and F. 0rskov. 1972. Polynucleotide sequence relat-edness among three groups of pathogenic Escher-ic hia colistrains. Infect. Immun. 6:308-315.

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7. Brenner, D. J., J. J. Farmer III, G. R. Fanning, A. G.Steigerwalt, P. Klykken, H. G. Wathen, F. W. Hickman,and W. H. Ewing. 1978. Deoxyribonucleic acid related-ness of Proteuis and Providencia species. Int. J. Syst.Bacteriol. 28:269-282.

8. Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, R. E.Weaver, J. C. Feeley, L. G. Cordes, H. W. Wilkinson, C.Patton, B. M. Thomason, and K. R. Lewallen Sasseville.1980. Legionella bhoetnanii sp. nov. and Legioiell/i c/ul-,tioffii sp. nov.: classification of two additional species ofLegiotnella associated with human pneumonia. Curr. Mi-crobiol. 4:111-116.

9. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergery'smanual of determinative bacteriology. 8th ed. The Wil-liams & Wilkins Co.. Baltimore.

10. Darland, G., and B. R. Davis. 1974. Biochemical andserological characterization of hydrogen sulfide-positivevariants of Escherichia coli. Center for Disease Control,Atlanta, Ga.

11. Edwards, P. R., and W. H. Ewing. 1972. Identification ofEnterobacterioceae. 3rd ed. Burgess Publishing Co.. Min-neapolis. Minn.

12. Farmer, J. J., III, G. R. Fanning, G. P. Huntley-Carter,B. Holmes, F. W. Hickman, C. Richard, and D. J. Bren-ner. 1981. Kl/uvver: a new (redefined) genus in Euitero-bacteriaceae. identification of K. ascorbota sp. nov.. andK. crvocrescens sp. nov. which occurs in clinical speci-mens. J. Clin. Microbiol. 13:919-933.

13. Hickman, F. W., and J. J. Farmer III. 1978. Sa/lmonellatvphi: identification. antibiograms, serology, and bacte-riophage typing. Am. J. Med. Technol. 44:1149-1159.

14. Lautrop, H., I. 0rskov, and K. Gaarslev. 1971. Hydrogensulphide producing variants of Esclherichi col/i. ActaPathol. Microbiol. Scand. Sect. B 79:641-650.

15. Layne, P., A. S. Hu, A. Balows, and B. R. Davis. 1971.Extrachromosomal nature of hydrogen sulfide productionin Escheric/hia coli. J. Bacteriol. 106:1029-1030.

16. Lesher, R. J., and W. H. Jones. 1978. Urease productionfrom clinical isolates of beta-hemolvtic Escherichija (/oli.J. Clin. Microbiol. 8:344-345.

17. Marmur, J., and P. Doty. 1962. Determination of the basecomposition of deoxyribonucleic acid from its thermaldenaturation temperature. J. Mol. Biol. 5:109-118.

18. 0rskov, I., F. 0rskov, W. J. Sojka, and J. M. Leach.1961. Simultaneous occurrence of E. coli B and L anti-gens in strains from diseased swine. Acta Pathol. Micro-biol. Scand. 53:404-422.

19. Sato, G., M. Asagi, C. Oka, N. Ishiguro, and N. Terakado.1978. Transmissible citrate-utilizing ability in Escherichiacoli isolated from pigeons. pigs and cattle. Microbiol.Immunol. 22:357-360.

20. Skerman, V. D. B., V. NMcGowan, and P. H. A. Sneath.1980. Approved lists of bacterial names. Int. J. Syst.Bacteriol. 30:225-420.

21. Wachsmuth, I. K., B. R. Davis, and S. D. Allen. 1979.Urealytic Escherichia( coli of human origin: serologic.epidemiologic, and genetic analysis. J. Clin. Microbiol.10:897-902.

22. Washington, J. A., II, and M. D. Maker. 1975. Unclassi-fied, lactose-fermenting. urease-producing members ofthe family Enterohocteriaceae resembling Es/cerichiaco/i. J. Clin. Microbiol. 2:70-71.

23. Washington, J. A., II, and J. A. Timm. 1976. Unclassi-fied. citrate positive member of the family Enterobaciteia-ceae resembling Eschzerichia coli. J. Clin. Microbiol.4:165-167.

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